The long-term objective of this project is the detailed analysis of a virulence-associated property: plasmid-mediated iron uptake, which plays an important role in the pathogenesis of many invasive diseases of man and domestic animals. Our model system is the pathogen Vibrio anguillarum which causes a terminal hemorrhagic septicemia. The features of this plasmid-mediated system of virulence makes it a very attractive model to study host-bacteria interactions. In the past granting periods, we characterized the essential components of this iron-uptake system: the siderophore anguibactin, the transport proteins FatA, B, C, and D as well as regulatory elements controlling their biosynthesis. The specific mechanisms by which these components govern the process of iron uptake, an thus virulence in this bacterium will be further elucidated by the proposed investigation. In the following, I will describe the specific aims to achieve these objectives: 1. The process of iron uptake in V. anguillarum is positively regulated at the transcriptional level by the action of the 110 kDa protein AngR and product(s) of the pJM1 TAF region which act synergistically. Therefore, we will analyze in detail the mechanism of positive regulation by the AngR protein and products of the TAF region as well as the function of AngR in the biosynthesis of the siderophore anguibactin. 2. Under iron-rich conditions, expression of the pJM1- mediated iron-uptake system is repressed by a chromosomally-encoded Fur protein and by plasmid-mediated antisense RNAs: RNAalpha which plays a role on the expression of iron transport protein genes and RNAbeta which controls the expression of AngR. We will, therefore, investigate the mechanism of action of these three repressor at the molecular level. 3. We have identified that athe fatA protein is the receptor for ferric anguibactin complexes, that fatB is a lipoprotein that shares domain homology with other periplasmic-binding proteins but is isolated in the membrane fraction, and that FatC and FatD are cytoplasmic integral membrane proteins. Therefore, we propose to precisely determine, by using gene fusions, mutant analysis, and ferric-siderophore binding studies, the localization of domains of these proteins to the various cellular compartments, to establish their hierarchy in iron transport. 4. We have subcloned specific regions of the pJM1 iron-uptake region and used complementation to identify anguibactin biosynthetic genes in the pJM1 plasmid and on the chromosome. We, therefore, propose in this granting period to complete the genetic and molecular dissection of the anguibactin biosynthetic pathway. In summary, our molecular studies should provide useful information in both the basic aspects of plasmid-mediated iron uptake as well as on its role as a bacterial virulence factor, by providing insight into the basic mechanisms of survival of this bacterium under conditions of iron starvation such as world be experienced in the circulatory system and tissues of the vertebrate host. An understanding of signal transduction in bacteria may also reveal new strategies for combating infectious disease. Results obtained in this system are thus directly applicable to other systems involved in pathogenesis.